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Effect of raspberry extract on wound healing

Effect of raspberry extract on wound healing The main purpose of this study was to investigate the effect of raspberry extract on wound healing and compare it with that of ellagic acid. The elimination of excess free radicals was the key to preventing wound inflammation; cellular antioxidation activity was evaluated using an oxidative stress damage cell model. Cell proliferation ability was measured using the WST-1 assay, and the migration capacity was determined using the wound scratch assay. A mouse wound model was used to verify the effect of raspberry extract on wound healing. The cellular antioxidant activity of the extract ((50.31±3.17) μg/mL) was slightly lower than that of ellagic acid ((44.59±2.38) μg/mL). The results of a cell proliferation assay showed that both raspberry extract and ellagic acid at 5 μg/mL could significantly (P<0.01) promote the proliferation of HaCaT cells. After culturing for 24 h and 48 h, the cell healing rates of the extract were (41.11±0.38) per cent and (68.88±2.51) per cent, respectively, whereas the corresponding rates of ellagic acid were (39.01±2.40) per cent and (70.33±0.89) per cent; hence, there were no significant differences between them (P>0.05). The wound areas of mice fed low, medium, and high doses of raspberry extract for 14 days were 1.66, 1.41, and 1.24  mm , respectively, which were significantly lower than that of the blank control group, 2.18  mm (P<0.05). These findings indicate that raspberry extract and ellagic acid exhibit similar antioxidant capacities and equivalent cell proliferation-promoting capabilities. In the mouse test, raspberry extract effectively promoted a reduction in wound area. This work demonstrates the potential of raspberry extract in wound healing, suggesting a promising application of raspberry resources in the fields of functional foods, cosmetics, and medicine. Keywords: Raspberry extract; ellagic acid; antioxidant activity; cell proliferation; wound healing. intervention treatments for ailments such as diabetes, inflammation, Introduction immunomodulatory disorders, and hyperlipidemia (Teng et al., 2017; Raspberry (Rubus idaeus L.) is a Rosaceae plant that is both a medi- Baby et al., 2018; Kowalska et al., 2019; Zhao et al., 2020). cine and food, and is mainly distributed in the temperate regions of Ellagic acid is widely distributed in various fruits and nuts the northern hemisphere (Giuffrè et  al., 2019). Raspberries contain (Vadhanam et  al., 2011). The chemical structure of ellagic acid is ellagic acid, salicylic acid, superoxide dismutase, polysaccharides, shown in Figure 1. The ellagic acid content in red raspberry has been anthocyanins, flavonoids, vitamins, and other active ingredients. They reported to be 119.8–323.5 mg/100 g (Bobinaitė et al., 2012). Ellagic are of high value as food, as well as in Chinese traditional medicine as acid exhibits strong antioxidant, antibacterial (Bobinaitė et  al., © The Author(s) 2021. Published by Oxford University Press on behalf of Zhejiang University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which per- mits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 2 W. J. Lu et al. from Beyotime (Shanghai, China). Kunming mice were ordered from SPF Biotechnology Co., Ltd. (Beijing, China). O OH Cell culture and animal feeding Human liver cancer SMMC-7721 cells were obtained from the HO OH Cell Bank of the Chinese Academy of Sciences (Beijing, China), and human immortalized epidermal HaCaT cells were obtained from Shengbo Biomedical Technology Co., Ltd. (Shanghai, China). Cell HO O culture was conducted in an incubator with a humidified atmosphere of 5 per cent CO at 37 °C. The SMMC-7721 cells were cultured in RPMI 1640 medium containing 10 per cent fetal bovine serum (FBS; ExCell Bio, Shanghai, China), whereas HaCaT cells were maintained Figure 1. Structural formula of ellagic acid. in DMEM. Both media contained 1 per cent penicillin and strepto- mycin mixture. 2013; Bobinaitė et al., 2016; Teng and Chen, 2019), and anticancer Twenty-five male mice at 4 weeks of age and and weighing effects (Khanduja et  al., 1999; Hussein and Khalifa, 2014; Aslan 18–20 g were used in this research. The mice were given commercial et al., 2020; Duan et al., 2020; Mansouri et al., 2020; Wang et al., food and water ad libitum. 2020). Yang et  al. (2019) found that an ointment made of pom- egranate seed ellagic acid could promote cell proliferation and heal Detection of SMMC-7721 cell antioxidation by skin burns and wounds. Ellagic acid at concentrations of 40 per cent and above can enhance the functional effect of pomegranate extract raspberry extract (Jurenka, 2008; Kunle et al., 2012). SMMC-7721 cells in logarithmic growth phase were collected and As the largest organ system of the human body, the skin protects adjusted to a concentration of 1×10 cells/mL in RPMI 1640 me- the body from external stimuli. However, loss of skin integrity caused dium containing 10 per cent FBS. Then, the cells were inoculated by injury or disease is common. Studies have shown that a high in a 96-well plate at 100 μL/well with three replicate wells for each amount of free radicals surround a wound (Milvy et al., 1973; Kuhn, group. After culturing for 24  h, the culture medium was removed, 2003). The elimination of free radicals can accelerate the disappear- and the cells were washed once with PBS. Sample solution (100 μL) ance of inflammation and promote wound healing. Epidermal cells diluted with serum-free culture medium and containing DCFH-DA play a key role in the process of wound healing, and also affect the probe was added to make the final concentration of DCFH-DA speed and quality of the healing (Parnell, 2013). In a previous work 25 μmol/L. After incubation at 37 °C for an additional hour, the cul- study of our laboratory, it was found that raspberry extract contains ture medium was discarded. The cells were washed 1–2 times with 539.30  mg/g ellagic acid. In addition to ellagic acid, raspberry ex- 100 μL PBS (37 °C); then, 100 μL of 20 μmol/L ABAP was added. tract contains 120.30 mg/g ellagic acid dimer, 98.30 mg/g ellagic acid For the blank and control groups, only fresh medium was added. glucose derivatives, and 242.10  mg/g other unknown compounds. Absorbance was measured every 5 min for 1 h at 485 nm excitation This is somewhat different from the composition of pomegranate and 538 nm emission, using a microplate reader. Three wells were extract (Satomi et al., 1993; Romani et al., 2012). Although the bio- used for each measurement. logical roles of ellagic acid have been studied, the influence of rasp- The area under the time–fluorescence intensity curve was cal- berry extract on wound healing remains to be elucidated. culated. The CAA values of different concentrations of ellagic acid The main aim of this study was to evaluate the effect of raspberry were determined according to Equation (1): extract on wound healing. Using ellagic acid as a control, cellular antioxidant activity (CAA), cell proliferation, and migration assays SA ´ (1) CAA=100− ×100 were performed. Finally, verification was done using mice tests. CA where ∫SA (sample area) represents the integrated area under the Materials and Methods curve for the sample fluorescence intensity versus time, whereas ∫CA (control area) represents the integrated area from the control curve. Chemicals and standards The median effective dose (EC ) was calculated based on the Ellagic acid (high-performance liquid chromatography grade), 50 median effect principle of log (fa/fu) and log (concentration), where 2′,7′-dichloro-di hydrofluorescein diacetate (DCFH-DA), and “fa” is the fraction affected and “fu” is the fraction unaffected by 2,2′-azobis-2-methyl-propanimidamide dihydrochloride (ABAP) the treatment. The EC values were expressed as means±standard were ordered from Sigma-Aldrich (St. Louis, MO, USA). Raspberry 50 deviation (SD). extract containing 539.30 mg/g ellagic acid, 120.30 mg/g ellagic acid dimer, 98.30 mg/g ellagic acid glucose derivative, and 242.10 mg/g Detection of HaCaT cell proliferation induced by other constituents was prepared and characterized in our laboratory. Roswell Park Memorial Institute (RPMI) 1640 medium was obtained raspberry extract from Hyclone Laboratories  Inc. (Logan,  UT, USA). Dulbecco’s HaCaT cells in the logarithmic phase were adjusted to a concen- Modified Eagle Medium (DMEM) was obtained from Gibco (Grand tration of 2×10 cells/mL in DMEM containing 10 per cent FBS. Island, NY, USA). Dimethyl sulfoxide (DMSO), pancreatin-EDTA, Then, the cells were inoculated in a 96-well plate at 100  μL/well and phosphate-buffered saline (PBS) were obtained from Solarbio with three replicates for each treatment. After 24  h of incubation, (Beijing, China). Recombinant human epidermal growth factor the culture medium was discarded, raspberry extract or ellagic acid- (rhEGF) was procured from PeproTech (Rocky Hill, NJ, USA). The supplemented medium was added, and the cell were cultured for WST-1 Cell Proliferation and Cytotoxicity Assay Kit was purchased another 24  h. Cell proliferation was determined using the WST-1 Effect of raspberry extract on wound healing 3 cell proliferation and cytotoxicity detection kit. The control group mice were fasted for 8  h after the operation, but were allowed to was treated without ellagic acid; the blank group was treated only drink water normally. The day of model building was recorded as with cell culture medium, and contained no cells. The OD values day 0. After 12 h of modeling, the mice were administered the test were measured at 450 nm on a microplate reader, and recorded as sample (0.2 mL/10 g) by oral gavage every day; the control group OD , OD , and OD . The cell proliferation rate was cal- was administered physiological saline. The wound condition was ob- experiment control blank culated according to Equation (2): served and the remaining area of the wound was calculated. OD −OD experiment blank Statistical analysis Cell proliferation rate (%) = ×100% OD −OD control blank Delimitation of regions of interest and quantification of struc- (2) tures were performed using built-in tools in Image J 1.8.0 image processing software. All the samples were processed and analyzed in Detection of HaCaT cell migration induced by triplicate. The values were expressed as means±SD. Statistical ana- raspberry extract lyses were performed using IBM SPSS Statistics version 17.0 (SPSS HaCaT cells (6×10 cells/mL) were inoculated on a cell culture plate Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) fol- and cultured for 24 h to cover 90 per cent of the well area. Then, lowed by a Student’s t-test was applied to determine the statistical a line was drawn with a 200 μL tip along a ruler. The cells were significance of differences between data. Differences with P<0.05 washed 2–3 times with PBS and then cultured with medium con- were considered statistically significant. taining 5 μg/mL of raspberry extract or ellagic acid. The blank group was not processed. The positive control group was cultured with a medium containing 3 ng/L rhEGF. Images were captured using an in- Results and Discussion verted microscope at 0, 24, and 48 h. Image J 1.8.0 image processing Cellular antioxidant activity of raspberry extract software (Joonas “Regalis” Rikkonen, Turku, Finland) was used to The antioxidant activity of raspberry extract was evaluated using calculate the area of the irregular figure at the scratch. The scratch the cellular antioxidant assay (Adom and Liu, 2005; Wolfe and Liu, closure rate at different time points was calculated according to 2007). In this assay, free radicals induced by ABAP are the main Equation (3): type of free radicals in the human body, resulting in a better simula- tion of the damage caused by peroxy free radicals (Niki, 1990). The S −S 0 t (3) Scratch healing rate (%) = ×100% S results of the cellular antioxidant assay for raspberry extract and ellagic acid are shown in Figure 2A and 2B, respectively. As indi- where S is the scratch area of the cell at 0 h, and S is the scratch 0 t cated, the fluorescence intensity of raspberry extract and ellagic acid area of the cell at t. increased steadily with the incubation time, whereas it gradually decreased with an increase in the concentrations (1–50  μg/mL). Measurement of wound healing area in mice This observation is consistent with the results of the study per- Mice were adapted in the laboratory for a week before the pro- formed by Katsunari et al. (2009) who found that the antioxidant cedures. Then, the mice were randomly separated into five groups, activity of an extract and ellagic acid was positively associated namely, blank control group, raspberry extract groups (low, medium, with the concentration. Both raspberry extract and ellagic acid in- and high doses: 20, 40, and 80  mg/kg, respectively), and rhEGF hibited the oxidation process of 2’,7’-dichlorofluorescein diacetate (5 μg/kg) group, each with 5 animals. After intraperitoneal injection (DCF) effectively (Hanneken et  al., 2006; Rüweler et  al., 2008), of 10 per cent chloral hydrate to anesthetize the mice, the back hair thus reducing the fluorescence intensity and showing good anti- was cut off, and the full-thickness skin was sampled 0.5  cm from oxidant capacity. The EC values were used to determine the anti- the midline of the back of the mice using a 4 mm skin sampler. The oxidant capacity. The concentration of an antioxidant is inversely A B 0μg/mL 0μg/mL 1μg/mL 1μg/mL 10 μg/mL 10 μg/mL 20 μg/mL 20 μg/mL 30 μg/mL 30 μg/mL 40 μg/mL 40 μg/mL 50 μg/mL 50 μg/mL 0 0 0102030405060 0102030405060 Time (min) Time (min) Figure 2. Kinetic curve of fluorescence intensity versus time. (A) is the experimental group of raspberry extract, and (B) is the experimental group of ellagic acid. Fluorescencevalue Fluorescence value 4 W. J. Lu et al. proportional to its antioxidant activity; that is, a small EC value shows the cell proliferation rates determined at different concen- indicates a strong antioxidant capacity. The EC values of the ex- trations of raspberry extract and ellagic acid. The cell proliferation tract and ellagic acid were calculated based on the regression equa- rate increased with the concentration. When the concentration of tion shown in Figure 3 and the dose–antioxidant median effect the extract was 0.63 μg/mL, the proliferation rate was significantly diagram shown in Figure 4. The EC values of raspberry extract (P<0.05) higher than that of the control group. Similarly, when the and ellagic acid were (50.31±3.17) μg/mL and (44.59±2.38) μg/mL, concentration of ellagic acid was 1.25 μg/mL, the cell proliferation respectively. The antioxidant activity of raspberry extract was rate was significantly higher than that of the control group (P<0.05). slightly lower than that of ellagic acid. Our previous study showed These findings indicate that the effect of raspberry extract on cell that ellagic acid accounted for 53 per cent of the raspberry extract proliferation was not limited by its ellagic acid content. The other contents (539.30 mg/g). The antioxidant activity of the extract was components present in the extract may have also contributed to the approximately 89 per cent that of ellagic acid, indicating that the cell proliferation. At 5.00 μg/mL, the cell proliferation rate of the other components present in the extract might also exert antioxi- two treatment groups was significantly (P<0.01) higher than that dant activity (Zheng et al., 2020), but not as strongly as the ellagic at lower concentrations, but there was no significant difference be- acid monomer. This could be plausibly explained by the fact that tween the groups (P>0.05). the ellagic acid dimer and ellagic acid glucose derivatives have large These results are in partial agreement with those of Mottola molecular weights and contain various hydroxyl groups, which can et  al. (2020), who reported that ellagic acid has a protective effect easily cause intramolecular or intermolecular hydrogen bond inter- on the proliferation of mammalian cells. In addition, this study dem- actions, and further change the conformation and affect the antioxi- onstrates that there is no obvious relationship between the antioxi- dant activity (Fogliani et al., 2005). dant activity of antioxidants and their cell proliferation-promoting ability, which is in contrast to the finding of Mendis et  al. (2005). This might be due to the difference in efficiency between the ellagic Effect of raspberry extract on HaCaT cell acid dimer and ellagic acid glucose derivative in raspberry extract, proliferation or because the antioxidant activity of the extract and ellagic acid The effects of raspberry extract and ellagic acid on the proliferation eliminated excessive free radicals that affect cell proliferation. In the of epidermal cells during wound healing were compared. Figure 5 process of wound healing, subsequent injury caused by free radicals A B 60 60 50 50 40 40 30 30 20 20 R =0.9835 R =09927 10 10 0 0 01020304050 01020304050 Concentration(μg/mL) Concentration(μg/mL) Figure 3. Effect of extract (A) and ellagic acid (B) at different concentrations on cellular antioxidant activity (CAA). A B 0.4 0.2 0.2 0.0 0.0 -0.2 -0.2 -0.4 -0.4 -0.6 -0.6 -0.8 -0.8 -1.0 -1.0 -1.2 Log(fa/fu)=0.8012*Log(conc)-1.3376 R=0.9110 Log(fa/fu)=0.7842*Log(conc)-1.3036 R=0.9844 -1.2 Log(fa/fu)=0.9617*Log(conc)-1.5538 R=0.9788 Log(fa/fu)=0.6880*Log(conc)-1.1782 R=0.9661 -1.4 Log(fa/fu)=0.7717*Log(conc)-1.2814 R=0.9261 Log(fa/fu)=0.6374*Log(conc)-1.1012 R=0.9555 -1.4 -1.6 -0.2 0.0 0.2 0.4 0.6 0.81.0 1.21.4 1.61.8 -0.2 0.0 0.20.4 0.6 0.8 1.01.2 1.41.6 1.8 Log(concentration,μg/mL) Log(concentration,μg/mL) Figure 4. Median effect of dose and antioxidant. (A) is the experimental group of raspberry extract, and (B) is the experimental group of ellagic acid. Log(fa/fu) CAA Log (fa/fu) CAA Effect of raspberry extract on wound healing 5 can be reduced to achieve favorable conditions for cell proliferation raspberry extract and ellagic acid to perform the treatment, which (Padma et al., 2014; Mehrzadi et al., 2019). had extremely significant effects on the proliferation of HaCaT cells compared with the control treatment. Figure 6 shows the prolifer- ation and migration of HaCaT cells cultured at 0, 24, and 48 h in Effect of raspberry extract on HaCaT cell the treatment, blank, and positive control groups. The scratch area scratch healing gradually decreased the culture time of HaCaT cells. A  significant HaCaT cell scratch assays were used to simulate cell growth and difference was found in the cell scratch areas at the different time phases of wound healing. This involved the intuitive manifest- periods (P<0.05). ation of cell proliferation and migration. We selected 5  μg/mL of Figure 7 shows that when the cells were cultured for 24  h, the scratch healing rate of the raspberry extract group (Y) was (41.11±1.92) per cent, which was not significantly different from that of the ellagic acid group (B) at (39.01±2.00) per cent (P>0.05). However, compared with that of the blank control  group (K) at (20.67±1.16) per cent, a very significant difference was found (P<0.01), indicating that raspberry extract and ellagic acid pro- moted cell proliferation and migration. This finding is consistent with the results of previous experiments on HaCaT cell proliferation, in which the cells were cultured for 48  h and the scratch healing rates of raspberry extract, ellagic acid, and rhEGF positive con- trol (R) groups were (68.88±1.84) per cent, (70.33±0.67) per cent, and (85.53±0.40) per cent, respectively, which were significantly higher than that of the blank group (34.21±1.30) per cent (P<0.01). During the culture, rhEGF was used as the positive control, and its effect on cells was significantly different from those of the blank, raspberry extract, and ellagic acid (P<0.01). Raspberry extract and ellagic acid promoted cell proliferation and migration, but signifi- cant differences existed in terms of their effects in comparison with that of rhEGF. The scratch results of HaCaT cells further confirmed the effect of raspberry extract and ellagic acid on the proliferation Figure 5. Effect of raspberry extract and ellagic acid on the proliferation rate of HaCaT cells. of HaCaT cells. * and ** above the bar indicate the significant differences Rens and Merks (2020) found that the interaction between cells compared with the control group (P<0.05 and P<0.01, respectively). and extracellular matrix has a certain impact on cell proliferation Figure 6. Effect of different treatments on HaCaT cell migration at different times. The culture times of subscript 1, 2, and 3 are 0, 24, and 48 h, respectively; 4 4 4 4 4 the scratch areas of K , K , K , B , B , B , Y , Y , Y , R , R , and R are: (31.98±1.22)×10 , (25.37±0.89)×10 , (21.04±1.21)×10 , (33.70±1.02)×10 , (20.56±1.29)×10 , 1 2 3 1 2 3 1 2 3 1 2 3 4 4 4 4 4 4 4 2 (10.00±0.08)×10 , (28.43±1.48)×10 , (16.74±1.41)×10 , (8.85±1.00)×10 , (30.39±1.03)×10 , (9.20±0.89)×10 , and (5.00±0.29)×10 μm , respectively. 6 W. J. Lu et al. and migration. Tang et al. (2015) found that ellagic acid can reduce 24 h the expression of inflammatory factors such as IL-1β and NLRP3. 48 h Therefore, the extract and ellagic acid may activate the expression ** of some genes directly or indirectly, promote the synthesis of related proteins, and increase cell proliferation efficiency or weaken the ## ## ** ** ** inhibitory effect of certain genes on cell proliferation, thereby pro- moting wound recovery (Jara et  al., 2020; Li et  al., 2020). Further work is needed to establish the relationship between the antioxi- dant and cell proliferation-promoting capacity, and to determine the ## ## underlying mechanisms for wound healing. ** ** ## ## in mice Skin wound healing is the process of gradual repair of skin tissue over time. After modeling, mice were continuously intervened for 14  days; the wound status of the mice is shown in Figure 8. The mice wounds did not show any adverse conditions such as infec- KBYR tion, pus accumulation, redness, or swelling. The skin wounds of Type raspberry extract gavage mice formed obvious dark-red hard scabs. After 12 days of intervention, the wound area was almost filled and Figure 7. Comparison of scratch healing rate of HaCaT cells among different the wounds healed well. After 14 days, the color around the wounds treatment groups. * and **above the bar indicate the significant differences became lighter, and the wounds gradually became normal tissue. compared with group K (P<0.05 and P<0.01, respectively); # and ## above the However, the wounds of mice in the rhEGF group did not achieve bar indicate the significant differences compared with group R (P<0.05 and P<0.01, respectively). the expected effect. It is speculated that rhEGF entered the mice and Figure 8. Wound healing in mice. K, blank control group; L, raspberry extract, low-dose group; M, raspberry extract, medium-dose group; H, raspberry extract, high-dose group; R, rhEGF positive control group. Scratch healing Rate (%) Effect of raspberry extract on wound healing 7 Table 1. Statistics of remaining wound area of mice Remaining wound area (mm ) Group type 0 day 4 days 8 days 12 days 14 days Blank control group (K) 13.15±0.49 9.81±0.55 6.61±0.44 2.97±0.72 2.18±0.23 Raspberry extract, low-dose group (L) 13.10±0.47 9.43±0.34 6.33±0.53 2.48±0.23 1.66±0.27* Raspberry extract, medium-dose group (M) 12.92±0.44 9.39±0.70* 5.94±0.39 2.34±0.16* 1.41±0.24** **# Raspberry extract, high-dose group (H) 13.05±0.42 8.73±0.67** 5.12±0.39 2.09±0.19** 1.24±0.11** rhEGF positive control group (R) 12.92±0.42 9.24±0.80 6.10±0.95 2.72±1.21 1.45±0.27** *Significant difference compared with group K (P<0.05); **significant difference compared with group K (P<0.01); #significant difference compared with group R (P<0.05). was decomposed by the digestive system, weakening the effect on Funding wound healing (Lee, 2002). This study was financially supported by the project grant from red rasp- Table 1 shows the changes in the wound area of mice. After berry high-efficiency cultivation technology integration and deep processing gavage with raspberry extract for 4  days, the remaining wound product development of Hebei Province, China (No.19226815D). This work area of mice was smaller than that of the blank control group (K). was also funded by the Modern Forestry Discipline Group (XK1008601519) The high-dose raspberry extract group (H) showed the highest ef- and the Food Processing Discipline Group (No.2021-05) of Hebei Agricultural University, China. fect on wound healing compared with the blank control group. The intervention effect was statistically significant (P <0.05), which suggested that raspberry extract promoted wound healing. Then, References as the dose of raspberry extract and intervention time increased, Adom,  K.  K., Liu,  R.  H. (2005). 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Effect of raspberry extract on wound healing

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Food Quality and Safety , Volume 5: 1 – Jun 23, 2021
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Oxford University Press
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© The Author(s) 2021. Published by Oxford University Press on behalf of Zhejiang University Press.
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2399-1399
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10.1093/fqsafe/fyab013
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Abstract

The main purpose of this study was to investigate the effect of raspberry extract on wound healing and compare it with that of ellagic acid. The elimination of excess free radicals was the key to preventing wound inflammation; cellular antioxidation activity was evaluated using an oxidative stress damage cell model. Cell proliferation ability was measured using the WST-1 assay, and the migration capacity was determined using the wound scratch assay. A mouse wound model was used to verify the effect of raspberry extract on wound healing. The cellular antioxidant activity of the extract ((50.31±3.17) μg/mL) was slightly lower than that of ellagic acid ((44.59±2.38) μg/mL). The results of a cell proliferation assay showed that both raspberry extract and ellagic acid at 5 μg/mL could significantly (P<0.01) promote the proliferation of HaCaT cells. After culturing for 24 h and 48 h, the cell healing rates of the extract were (41.11±0.38) per cent and (68.88±2.51) per cent, respectively, whereas the corresponding rates of ellagic acid were (39.01±2.40) per cent and (70.33±0.89) per cent; hence, there were no significant differences between them (P>0.05). The wound areas of mice fed low, medium, and high doses of raspberry extract for 14 days were 1.66, 1.41, and 1.24  mm , respectively, which were significantly lower than that of the blank control group, 2.18  mm (P<0.05). These findings indicate that raspberry extract and ellagic acid exhibit similar antioxidant capacities and equivalent cell proliferation-promoting capabilities. In the mouse test, raspberry extract effectively promoted a reduction in wound area. This work demonstrates the potential of raspberry extract in wound healing, suggesting a promising application of raspberry resources in the fields of functional foods, cosmetics, and medicine. Keywords: Raspberry extract; ellagic acid; antioxidant activity; cell proliferation; wound healing. intervention treatments for ailments such as diabetes, inflammation, Introduction immunomodulatory disorders, and hyperlipidemia (Teng et al., 2017; Raspberry (Rubus idaeus L.) is a Rosaceae plant that is both a medi- Baby et al., 2018; Kowalska et al., 2019; Zhao et al., 2020). cine and food, and is mainly distributed in the temperate regions of Ellagic acid is widely distributed in various fruits and nuts the northern hemisphere (Giuffrè et  al., 2019). Raspberries contain (Vadhanam et  al., 2011). The chemical structure of ellagic acid is ellagic acid, salicylic acid, superoxide dismutase, polysaccharides, shown in Figure 1. The ellagic acid content in red raspberry has been anthocyanins, flavonoids, vitamins, and other active ingredients. They reported to be 119.8–323.5 mg/100 g (Bobinaitė et al., 2012). Ellagic are of high value as food, as well as in Chinese traditional medicine as acid exhibits strong antioxidant, antibacterial (Bobinaitė et  al., © The Author(s) 2021. Published by Oxford University Press on behalf of Zhejiang University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which per- mits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 2 W. J. Lu et al. from Beyotime (Shanghai, China). Kunming mice were ordered from SPF Biotechnology Co., Ltd. (Beijing, China). O OH Cell culture and animal feeding Human liver cancer SMMC-7721 cells were obtained from the HO OH Cell Bank of the Chinese Academy of Sciences (Beijing, China), and human immortalized epidermal HaCaT cells were obtained from Shengbo Biomedical Technology Co., Ltd. (Shanghai, China). Cell HO O culture was conducted in an incubator with a humidified atmosphere of 5 per cent CO at 37 °C. The SMMC-7721 cells were cultured in RPMI 1640 medium containing 10 per cent fetal bovine serum (FBS; ExCell Bio, Shanghai, China), whereas HaCaT cells were maintained Figure 1. Structural formula of ellagic acid. in DMEM. Both media contained 1 per cent penicillin and strepto- mycin mixture. 2013; Bobinaitė et al., 2016; Teng and Chen, 2019), and anticancer Twenty-five male mice at 4 weeks of age and and weighing effects (Khanduja et  al., 1999; Hussein and Khalifa, 2014; Aslan 18–20 g were used in this research. The mice were given commercial et al., 2020; Duan et al., 2020; Mansouri et al., 2020; Wang et al., food and water ad libitum. 2020). Yang et  al. (2019) found that an ointment made of pom- egranate seed ellagic acid could promote cell proliferation and heal Detection of SMMC-7721 cell antioxidation by skin burns and wounds. Ellagic acid at concentrations of 40 per cent and above can enhance the functional effect of pomegranate extract raspberry extract (Jurenka, 2008; Kunle et al., 2012). SMMC-7721 cells in logarithmic growth phase were collected and As the largest organ system of the human body, the skin protects adjusted to a concentration of 1×10 cells/mL in RPMI 1640 me- the body from external stimuli. However, loss of skin integrity caused dium containing 10 per cent FBS. Then, the cells were inoculated by injury or disease is common. Studies have shown that a high in a 96-well plate at 100 μL/well with three replicate wells for each amount of free radicals surround a wound (Milvy et al., 1973; Kuhn, group. After culturing for 24  h, the culture medium was removed, 2003). The elimination of free radicals can accelerate the disappear- and the cells were washed once with PBS. Sample solution (100 μL) ance of inflammation and promote wound healing. Epidermal cells diluted with serum-free culture medium and containing DCFH-DA play a key role in the process of wound healing, and also affect the probe was added to make the final concentration of DCFH-DA speed and quality of the healing (Parnell, 2013). In a previous work 25 μmol/L. After incubation at 37 °C for an additional hour, the cul- study of our laboratory, it was found that raspberry extract contains ture medium was discarded. The cells were washed 1–2 times with 539.30  mg/g ellagic acid. In addition to ellagic acid, raspberry ex- 100 μL PBS (37 °C); then, 100 μL of 20 μmol/L ABAP was added. tract contains 120.30 mg/g ellagic acid dimer, 98.30 mg/g ellagic acid For the blank and control groups, only fresh medium was added. glucose derivatives, and 242.10  mg/g other unknown compounds. Absorbance was measured every 5 min for 1 h at 485 nm excitation This is somewhat different from the composition of pomegranate and 538 nm emission, using a microplate reader. Three wells were extract (Satomi et al., 1993; Romani et al., 2012). Although the bio- used for each measurement. logical roles of ellagic acid have been studied, the influence of rasp- The area under the time–fluorescence intensity curve was cal- berry extract on wound healing remains to be elucidated. culated. The CAA values of different concentrations of ellagic acid The main aim of this study was to evaluate the effect of raspberry were determined according to Equation (1): extract on wound healing. Using ellagic acid as a control, cellular antioxidant activity (CAA), cell proliferation, and migration assays SA ´ (1) CAA=100− ×100 were performed. Finally, verification was done using mice tests. CA where ∫SA (sample area) represents the integrated area under the Materials and Methods curve for the sample fluorescence intensity versus time, whereas ∫CA (control area) represents the integrated area from the control curve. Chemicals and standards The median effective dose (EC ) was calculated based on the Ellagic acid (high-performance liquid chromatography grade), 50 median effect principle of log (fa/fu) and log (concentration), where 2′,7′-dichloro-di hydrofluorescein diacetate (DCFH-DA), and “fa” is the fraction affected and “fu” is the fraction unaffected by 2,2′-azobis-2-methyl-propanimidamide dihydrochloride (ABAP) the treatment. The EC values were expressed as means±standard were ordered from Sigma-Aldrich (St. Louis, MO, USA). Raspberry 50 deviation (SD). extract containing 539.30 mg/g ellagic acid, 120.30 mg/g ellagic acid dimer, 98.30 mg/g ellagic acid glucose derivative, and 242.10 mg/g Detection of HaCaT cell proliferation induced by other constituents was prepared and characterized in our laboratory. Roswell Park Memorial Institute (RPMI) 1640 medium was obtained raspberry extract from Hyclone Laboratories  Inc. (Logan,  UT, USA). Dulbecco’s HaCaT cells in the logarithmic phase were adjusted to a concen- Modified Eagle Medium (DMEM) was obtained from Gibco (Grand tration of 2×10 cells/mL in DMEM containing 10 per cent FBS. Island, NY, USA). Dimethyl sulfoxide (DMSO), pancreatin-EDTA, Then, the cells were inoculated in a 96-well plate at 100  μL/well and phosphate-buffered saline (PBS) were obtained from Solarbio with three replicates for each treatment. After 24  h of incubation, (Beijing, China). Recombinant human epidermal growth factor the culture medium was discarded, raspberry extract or ellagic acid- (rhEGF) was procured from PeproTech (Rocky Hill, NJ, USA). The supplemented medium was added, and the cell were cultured for WST-1 Cell Proliferation and Cytotoxicity Assay Kit was purchased another 24  h. Cell proliferation was determined using the WST-1 Effect of raspberry extract on wound healing 3 cell proliferation and cytotoxicity detection kit. The control group mice were fasted for 8  h after the operation, but were allowed to was treated without ellagic acid; the blank group was treated only drink water normally. The day of model building was recorded as with cell culture medium, and contained no cells. The OD values day 0. After 12 h of modeling, the mice were administered the test were measured at 450 nm on a microplate reader, and recorded as sample (0.2 mL/10 g) by oral gavage every day; the control group OD , OD , and OD . The cell proliferation rate was cal- was administered physiological saline. The wound condition was ob- experiment control blank culated according to Equation (2): served and the remaining area of the wound was calculated. OD −OD experiment blank Statistical analysis Cell proliferation rate (%) = ×100% OD −OD control blank Delimitation of regions of interest and quantification of struc- (2) tures were performed using built-in tools in Image J 1.8.0 image processing software. All the samples were processed and analyzed in Detection of HaCaT cell migration induced by triplicate. The values were expressed as means±SD. Statistical ana- raspberry extract lyses were performed using IBM SPSS Statistics version 17.0 (SPSS HaCaT cells (6×10 cells/mL) were inoculated on a cell culture plate Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) fol- and cultured for 24 h to cover 90 per cent of the well area. Then, lowed by a Student’s t-test was applied to determine the statistical a line was drawn with a 200 μL tip along a ruler. The cells were significance of differences between data. Differences with P<0.05 washed 2–3 times with PBS and then cultured with medium con- were considered statistically significant. taining 5 μg/mL of raspberry extract or ellagic acid. The blank group was not processed. The positive control group was cultured with a medium containing 3 ng/L rhEGF. Images were captured using an in- Results and Discussion verted microscope at 0, 24, and 48 h. Image J 1.8.0 image processing Cellular antioxidant activity of raspberry extract software (Joonas “Regalis” Rikkonen, Turku, Finland) was used to The antioxidant activity of raspberry extract was evaluated using calculate the area of the irregular figure at the scratch. The scratch the cellular antioxidant assay (Adom and Liu, 2005; Wolfe and Liu, closure rate at different time points was calculated according to 2007). In this assay, free radicals induced by ABAP are the main Equation (3): type of free radicals in the human body, resulting in a better simula- tion of the damage caused by peroxy free radicals (Niki, 1990). The S −S 0 t (3) Scratch healing rate (%) = ×100% S results of the cellular antioxidant assay for raspberry extract and ellagic acid are shown in Figure 2A and 2B, respectively. As indi- where S is the scratch area of the cell at 0 h, and S is the scratch 0 t cated, the fluorescence intensity of raspberry extract and ellagic acid area of the cell at t. increased steadily with the incubation time, whereas it gradually decreased with an increase in the concentrations (1–50  μg/mL). Measurement of wound healing area in mice This observation is consistent with the results of the study per- Mice were adapted in the laboratory for a week before the pro- formed by Katsunari et al. (2009) who found that the antioxidant cedures. Then, the mice were randomly separated into five groups, activity of an extract and ellagic acid was positively associated namely, blank control group, raspberry extract groups (low, medium, with the concentration. Both raspberry extract and ellagic acid in- and high doses: 20, 40, and 80  mg/kg, respectively), and rhEGF hibited the oxidation process of 2’,7’-dichlorofluorescein diacetate (5 μg/kg) group, each with 5 animals. After intraperitoneal injection (DCF) effectively (Hanneken et  al., 2006; Rüweler et  al., 2008), of 10 per cent chloral hydrate to anesthetize the mice, the back hair thus reducing the fluorescence intensity and showing good anti- was cut off, and the full-thickness skin was sampled 0.5  cm from oxidant capacity. The EC values were used to determine the anti- the midline of the back of the mice using a 4 mm skin sampler. The oxidant capacity. The concentration of an antioxidant is inversely A B 0μg/mL 0μg/mL 1μg/mL 1μg/mL 10 μg/mL 10 μg/mL 20 μg/mL 20 μg/mL 30 μg/mL 30 μg/mL 40 μg/mL 40 μg/mL 50 μg/mL 50 μg/mL 0 0 0102030405060 0102030405060 Time (min) Time (min) Figure 2. Kinetic curve of fluorescence intensity versus time. (A) is the experimental group of raspberry extract, and (B) is the experimental group of ellagic acid. Fluorescencevalue Fluorescence value 4 W. J. Lu et al. proportional to its antioxidant activity; that is, a small EC value shows the cell proliferation rates determined at different concen- indicates a strong antioxidant capacity. The EC values of the ex- trations of raspberry extract and ellagic acid. The cell proliferation tract and ellagic acid were calculated based on the regression equa- rate increased with the concentration. When the concentration of tion shown in Figure 3 and the dose–antioxidant median effect the extract was 0.63 μg/mL, the proliferation rate was significantly diagram shown in Figure 4. The EC values of raspberry extract (P<0.05) higher than that of the control group. Similarly, when the and ellagic acid were (50.31±3.17) μg/mL and (44.59±2.38) μg/mL, concentration of ellagic acid was 1.25 μg/mL, the cell proliferation respectively. The antioxidant activity of raspberry extract was rate was significantly higher than that of the control group (P<0.05). slightly lower than that of ellagic acid. Our previous study showed These findings indicate that the effect of raspberry extract on cell that ellagic acid accounted for 53 per cent of the raspberry extract proliferation was not limited by its ellagic acid content. The other contents (539.30 mg/g). The antioxidant activity of the extract was components present in the extract may have also contributed to the approximately 89 per cent that of ellagic acid, indicating that the cell proliferation. At 5.00 μg/mL, the cell proliferation rate of the other components present in the extract might also exert antioxi- two treatment groups was significantly (P<0.01) higher than that dant activity (Zheng et al., 2020), but not as strongly as the ellagic at lower concentrations, but there was no significant difference be- acid monomer. This could be plausibly explained by the fact that tween the groups (P>0.05). the ellagic acid dimer and ellagic acid glucose derivatives have large These results are in partial agreement with those of Mottola molecular weights and contain various hydroxyl groups, which can et  al. (2020), who reported that ellagic acid has a protective effect easily cause intramolecular or intermolecular hydrogen bond inter- on the proliferation of mammalian cells. In addition, this study dem- actions, and further change the conformation and affect the antioxi- onstrates that there is no obvious relationship between the antioxi- dant activity (Fogliani et al., 2005). dant activity of antioxidants and their cell proliferation-promoting ability, which is in contrast to the finding of Mendis et  al. (2005). This might be due to the difference in efficiency between the ellagic Effect of raspberry extract on HaCaT cell acid dimer and ellagic acid glucose derivative in raspberry extract, proliferation or because the antioxidant activity of the extract and ellagic acid The effects of raspberry extract and ellagic acid on the proliferation eliminated excessive free radicals that affect cell proliferation. In the of epidermal cells during wound healing were compared. Figure 5 process of wound healing, subsequent injury caused by free radicals A B 60 60 50 50 40 40 30 30 20 20 R =0.9835 R =09927 10 10 0 0 01020304050 01020304050 Concentration(μg/mL) Concentration(μg/mL) Figure 3. Effect of extract (A) and ellagic acid (B) at different concentrations on cellular antioxidant activity (CAA). A B 0.4 0.2 0.2 0.0 0.0 -0.2 -0.2 -0.4 -0.4 -0.6 -0.6 -0.8 -0.8 -1.0 -1.0 -1.2 Log(fa/fu)=0.8012*Log(conc)-1.3376 R=0.9110 Log(fa/fu)=0.7842*Log(conc)-1.3036 R=0.9844 -1.2 Log(fa/fu)=0.9617*Log(conc)-1.5538 R=0.9788 Log(fa/fu)=0.6880*Log(conc)-1.1782 R=0.9661 -1.4 Log(fa/fu)=0.7717*Log(conc)-1.2814 R=0.9261 Log(fa/fu)=0.6374*Log(conc)-1.1012 R=0.9555 -1.4 -1.6 -0.2 0.0 0.2 0.4 0.6 0.81.0 1.21.4 1.61.8 -0.2 0.0 0.20.4 0.6 0.8 1.01.2 1.41.6 1.8 Log(concentration,μg/mL) Log(concentration,μg/mL) Figure 4. Median effect of dose and antioxidant. (A) is the experimental group of raspberry extract, and (B) is the experimental group of ellagic acid. Log(fa/fu) CAA Log (fa/fu) CAA Effect of raspberry extract on wound healing 5 can be reduced to achieve favorable conditions for cell proliferation raspberry extract and ellagic acid to perform the treatment, which (Padma et al., 2014; Mehrzadi et al., 2019). had extremely significant effects on the proliferation of HaCaT cells compared with the control treatment. Figure 6 shows the prolifer- ation and migration of HaCaT cells cultured at 0, 24, and 48 h in Effect of raspberry extract on HaCaT cell the treatment, blank, and positive control groups. The scratch area scratch healing gradually decreased the culture time of HaCaT cells. A  significant HaCaT cell scratch assays were used to simulate cell growth and difference was found in the cell scratch areas at the different time phases of wound healing. This involved the intuitive manifest- periods (P<0.05). ation of cell proliferation and migration. We selected 5  μg/mL of Figure 7 shows that when the cells were cultured for 24  h, the scratch healing rate of the raspberry extract group (Y) was (41.11±1.92) per cent, which was not significantly different from that of the ellagic acid group (B) at (39.01±2.00) per cent (P>0.05). However, compared with that of the blank control  group (K) at (20.67±1.16) per cent, a very significant difference was found (P<0.01), indicating that raspberry extract and ellagic acid pro- moted cell proliferation and migration. This finding is consistent with the results of previous experiments on HaCaT cell proliferation, in which the cells were cultured for 48  h and the scratch healing rates of raspberry extract, ellagic acid, and rhEGF positive con- trol (R) groups were (68.88±1.84) per cent, (70.33±0.67) per cent, and (85.53±0.40) per cent, respectively, which were significantly higher than that of the blank group (34.21±1.30) per cent (P<0.01). During the culture, rhEGF was used as the positive control, and its effect on cells was significantly different from those of the blank, raspberry extract, and ellagic acid (P<0.01). Raspberry extract and ellagic acid promoted cell proliferation and migration, but signifi- cant differences existed in terms of their effects in comparison with that of rhEGF. The scratch results of HaCaT cells further confirmed the effect of raspberry extract and ellagic acid on the proliferation Figure 5. Effect of raspberry extract and ellagic acid on the proliferation rate of HaCaT cells. of HaCaT cells. * and ** above the bar indicate the significant differences Rens and Merks (2020) found that the interaction between cells compared with the control group (P<0.05 and P<0.01, respectively). and extracellular matrix has a certain impact on cell proliferation Figure 6. Effect of different treatments on HaCaT cell migration at different times. The culture times of subscript 1, 2, and 3 are 0, 24, and 48 h, respectively; 4 4 4 4 4 the scratch areas of K , K , K , B , B , B , Y , Y , Y , R , R , and R are: (31.98±1.22)×10 , (25.37±0.89)×10 , (21.04±1.21)×10 , (33.70±1.02)×10 , (20.56±1.29)×10 , 1 2 3 1 2 3 1 2 3 1 2 3 4 4 4 4 4 4 4 2 (10.00±0.08)×10 , (28.43±1.48)×10 , (16.74±1.41)×10 , (8.85±1.00)×10 , (30.39±1.03)×10 , (9.20±0.89)×10 , and (5.00±0.29)×10 μm , respectively. 6 W. J. Lu et al. and migration. Tang et al. (2015) found that ellagic acid can reduce 24 h the expression of inflammatory factors such as IL-1β and NLRP3. 48 h Therefore, the extract and ellagic acid may activate the expression ** of some genes directly or indirectly, promote the synthesis of related proteins, and increase cell proliferation efficiency or weaken the ## ## ** ** ** inhibitory effect of certain genes on cell proliferation, thereby pro- moting wound recovery (Jara et  al., 2020; Li et  al., 2020). Further work is needed to establish the relationship between the antioxi- dant and cell proliferation-promoting capacity, and to determine the ## ## underlying mechanisms for wound healing. ** ** ## ## in mice Skin wound healing is the process of gradual repair of skin tissue over time. After modeling, mice were continuously intervened for 14  days; the wound status of the mice is shown in Figure 8. The mice wounds did not show any adverse conditions such as infec- KBYR tion, pus accumulation, redness, or swelling. The skin wounds of Type raspberry extract gavage mice formed obvious dark-red hard scabs. After 12 days of intervention, the wound area was almost filled and Figure 7. Comparison of scratch healing rate of HaCaT cells among different the wounds healed well. After 14 days, the color around the wounds treatment groups. * and **above the bar indicate the significant differences became lighter, and the wounds gradually became normal tissue. compared with group K (P<0.05 and P<0.01, respectively); # and ## above the However, the wounds of mice in the rhEGF group did not achieve bar indicate the significant differences compared with group R (P<0.05 and P<0.01, respectively). the expected effect. It is speculated that rhEGF entered the mice and Figure 8. Wound healing in mice. K, blank control group; L, raspberry extract, low-dose group; M, raspberry extract, medium-dose group; H, raspberry extract, high-dose group; R, rhEGF positive control group. Scratch healing Rate (%) Effect of raspberry extract on wound healing 7 Table 1. Statistics of remaining wound area of mice Remaining wound area (mm ) Group type 0 day 4 days 8 days 12 days 14 days Blank control group (K) 13.15±0.49 9.81±0.55 6.61±0.44 2.97±0.72 2.18±0.23 Raspberry extract, low-dose group (L) 13.10±0.47 9.43±0.34 6.33±0.53 2.48±0.23 1.66±0.27* Raspberry extract, medium-dose group (M) 12.92±0.44 9.39±0.70* 5.94±0.39 2.34±0.16* 1.41±0.24** **# Raspberry extract, high-dose group (H) 13.05±0.42 8.73±0.67** 5.12±0.39 2.09±0.19** 1.24±0.11** rhEGF positive control group (R) 12.92±0.42 9.24±0.80 6.10±0.95 2.72±1.21 1.45±0.27** *Significant difference compared with group K (P<0.05); **significant difference compared with group K (P<0.01); #significant difference compared with group R (P<0.05). was decomposed by the digestive system, weakening the effect on Funding wound healing (Lee, 2002). This study was financially supported by the project grant from red rasp- Table 1 shows the changes in the wound area of mice. 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Journal

Food Quality and Safety – Oxford University Press

Published: Jun 23, 2021

Keywords: Raspberry extract; ellagic acid; antioxidant activity; cell proliferation; wound healing

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